EX VIVO AND IN VITRO MODELS TO STUDY THE EFFECTS OF HYPOXIA AND INFLAMMATION ON HUMAN PLACENTAL MITOCHONDRIA

Intrauterine Growth Restriction (IUGR) is a pregnancy-related pathology characterized by a placental insufficiency phenotype and a multifactorial etiology that still needs to be completely clarified. IUGR is associated with increased risk of maternal and neonatal perinatal mortality and morbidity and a tendency to develop cardiovascular and metabolic pathologies in the adulthood. A deeper knowledge of the alterations occurring in IUGR has therefore become essential to find therapeutic tools to prevent fetal, neonatal and future adult complications. A specific placental phenotype has been associated with IUGR, characterized by placentation defects, altered transport of oxygen and nutrients to the fetus, impaired mitochondria content and increased oxidative stress (OxS). Mitochondria (mt) are eukaryotic ubiquitous organelles whose number range from hundreds to thousands of copies per cell. As they are the fuel stations of all cells, more than 95% of ATP is synthesized in these organelles Besides this well-known function, many essential pathways involve mitochondria, such as mt biogenesis. Mt biogenesis is a complex of mechanisms needed to mitochondria ex-novo creation: mt DNA duplication and translation of mt factors controlling the transcription machinery that produce all respiratory chain complexes (RCC). IUGR hypoxic features, and the consequent higher OxS, affect mitochondria as showed by in vivo models increased mt oxygen consumption trigger by hypoxia or in vitro downregulation of mt biogenesis. The aim of this study was to investigate, by ex vivo experiments and in vitro models, different types of placental cells to deeper characterize the placental insufficiency features of IUGR, with specific attention to the consequences of its hypoxic environment. IUGR and physiological placenta bioenergetics were first examined, by analyzing both mitochondrial (mt) content and function in whole placental tissue and in several placental cell types (cytotrophoblast and mesenchymal stromal cells). Mt DNA content resulted higher in IUGR placentas compared to controls, as well as NRF1 (biogenesis activator) mRNA levels. Oppositely, both mtDNA and NRF1 expression levels were significantly lower in cytotrophoblast cells isolated from IUGR placentas compared to controls. The observed divergence between placental tissue and cytotrophoblast cells may suggest that other placental cell types (e.g. syncytiotrophoblast, endothelial cells and mesenchymal stromal cells), that are subjected to different oxygen - and consequently oxidative stress - levels may be responsible for the mt content increase in the whole placental tissue. Moreover, a different exposure to progesterone may also explain this mt content divergence, since progesterone, regulating mt biogenesis, is produced by syncytio but not in cytotrophoblast cells. In IUGR cytotrophoblast cells, respiratory chain complexes (RCC) showed lower, though not significantly, gene expression levels and no differences in their protein expression compared to controls. In contrast, mt bioenergetics - represented by cellular O2 consumption - was higher in IUGR versus controls, especially in more severe IUGR cases. Thus, despite the protein content of RCC was not altered, their activity was significantly increased in IUGR cytotrophoblast cells, possibly due to a more efficient RCC assembly. Finally, as O2 consumption resulted inversely correlated to mtDNA in cytotrophoblast cells, a functional (respiration) compensatory effect to the decreased mitochondrial content might be hypothesized. Estrogen-Related Receptor (ERRγ) is a very interesting transcriptional factor involved both in mt biogenesis and function and in estradiol production (through CYP19 aromatase up-regulation). ERRγ and CYP19 mRNA levels were therefore analyzed, for the first time in human IUGR placentas. In whole placental tissue CYP19 showed higher expression in IUGR compared to controls, progressively increasing with IUGR severity. Higher ERRγ expression in IUGR cases was also found, though not significantly. These data are consistent with mtDNA and NRF1 results, thus confirming altered mt biogenesis and content in IUGR and strengthening the hypothesis of a restore attempt made through the stimulation of mt biogenesis. An additional effect of ERRγ increase is CYP19 upregulation. The observed higher CYP19 expression may indicate a protective mechanism exerted through estradiol against oxidative stress. Opposite to their placental tissue expression, ERRγ levels in cytotrophoblast cells significantly decreased in the IUGR group compared to controls. This is consistent with literature evidences of O2-dependent ERRγ gene expression in trophoblast cells. As well as for mt DNA and NRF1 levels, other cell types could be responsible for ERRγ increase in the whole placental tissue. CYP19 expression was not significantly different between IUGR and controls in cytotrophoblast cells, though it positively correlates with ERRγ levels, but low CYP19 levels are reported for cytotrophoblast cells, and this might complicate the detection of any difference. Interestingly, a significant positive correlation linked maternal BMI and expression of both ERRγ and CYP19 genes (in whole placental tissue: positive trend/cytotrophoblast cells: negative trend). An estradiol-dependent regulation of leptin production through ER (Estrogen Receptor) – ERR is known. Leptin, an anti-obesity hormone produced also by placenta, increase during. The future measure of plasmatic levels of both leptin and 17β estradiol in maternal blood will verify this speculation. Then in vitro experiments were performed to assess possible biomolecular mechanisms regulating mithocondrial content in Intrauterine Growth Restriction, by culturing primary placental cells under normal oxygen conditions and hypoxia, a typical feature of IUGR. Fluctuations in placental oxygen concentration may generate oxidative stress (OxS), that is enhanced in Intrauterine Growth Restriction condition. As mitochondria are the major producers of intracellular reactive oxygen (O2) species through free radicals generated by the mt oxidative phosphorylation, altered intrauterine O2 conditions might affect mt DNA content and function, leading to increased oxidative stress in IUGR placental cells. Using trophoblast primary cell lines could help to understand O2 conditions that placentas may be exposed to in IUGR pregnancy. Exposure of trophoblast cultures to hypoxia is an in vitro model commonly used in the last few years. Preliminary data from performed experiments show that the oxygen lack in cytotrophoblast cells leads to increased mt DNA levels. The evidence that O2 levels may regulate mt biogenesis in cytotrophoblast cells highlights their deep sensitivity to O2 conditions. However, further data are needed to confirm these preliminary results, also considering the implied difficulties in adapting the primary cytotrophoblast cultures, very sensitive to O2 concentration, to an in vitro model. A future goal will be reproduce particularly hypoxia/re-oxygenation intervals characterizing placental insufficiency and generating OxS and measuring cell apoptosis levels and autophagy markers (e.g. TNF-α, p53, caspases). Finally, in vitro experiments were performed to isolate and characterized p-MSCs from physiological and affected by IUGR placentas. p-MSCs have never been investigated before in IUGR pregnancies, but their role have been recently studied in preeclamptic placentas. PE p-MSCs show pro-inflammatory and anti-angiogenic features, that may result in abnormal placental development. In the performed p-MSCs cultures, mesenchymal markers enrichment and multipotent differentiation abilities confirm the successful isolation and selection of a mesenchymal stromal cell from placental membranes and basal disc of both physiological and IUGR placentas. As attested by flow cytometry data, the p-MSC population is earlier selected in IUGR placentas: this faster selection might represent a compensatory mechanism to metabolic alterations occurring in IUGR placental cells and/or to the adverse IUGR placental environment. During placenta development, the lower proliferation rate characterizing IUGR pMSCs could impair the primary villi formation and consequently trophoblast development, since MSCs both serve as structural of trophoblast cells. Moreover, IUGR p-MSCs population display lower endothelial and higher adipogenic differentiation potentials compared to controls. During pregnancy, pMSCs usually contribute to both vasculogenesis and angiogenesis Interestingly, several studies report some alterations in maternal and fetal endothelial progenitor or in the angiogenic capacity of IUGR placental cells. Opposite to endothelial differentiation ability, the adipogenic potential in pMSCs from IUGR is increased compared to controls: as these changes are evident early in life, the predisposition to obesity may be programmed in utero. To further characterize IUGR pMSCs, their mitochondrial (mt) content was investigated by measuring NRF1 and Respiratory Chain UQCRC1 and COX4I1 gene expression levels. Mesenchymal stem cell metabolism is known to be mainly anaerobic, with a shift towards an aerobic mitochondrial metabolism reported during differentiation. Interestingly, p-MSCs cultured with no differentiating medium present a trend towards higher NRF1, UQCRC1 and COX4I1 expression levels in IUGR basal disc samples compared to controls and higher COX4I1 levels in IUGR placental membranes; these differences are not statistically significant likely because of the low sample number. Nevertheless, they might account for metabolic alterations in IUGR p-MSCs, showing a possible shift to aerobic metabolism, with the loss of the metabolic characteristics that are typical of multipotent and undifferentiated cells. The different gestational age between cases and controls, typical of all IUGR versus term-placentas studies, is a possible limit that associate all the performed experiments. However, any significant correlation between gestational age (ge) and the O2 consumption of CIV (which presents the highest significance between IUGR and controls), ge and mt DNA levels, ge and ERRy/CYP19 expression, ge and p-MSCs. CYP19 gene expression have been analyzed assuming that it may represent an index of aromatase content in placental tissue. However, post-translational modifications (glycosylation and phosphorylation) may occur, affecting its functional activity. Finally, a potential limitation of placental mesenchymal stromal cells is that the analysis was performed on IUGR placentas at delivery, whereas placental abnormal development of IUGR pathology is supposed to start already at the beginning of placentation. Taken together, reported data highlight mitochondrial alterations occurring in placentas of Intrauterine Growth Restricted pregnancies, through ex vivo and in vitro approaches. These results shed genuine new data into the complex physiology of placental oxygenation in IUGR fetuses. Mitochondrial content is higher in IUGR total placental tissue compared with normal pregnancies at term. This difference is reversed in cytotrophoblast cells of IUGR fetuses, which instead present higher mitochondrial functionality. These findings suggest different mitochondrial features depending on the placental cell lineage. Indeed, our results on placental Mesenchymal Stromal Cells, showed higher levels of genes accounting for mitohcondrial content and function. The increased placental O2 consumption by placental tissue may represent a limiting step in fetal growth restriction, preventing adequate O2 delivery to the fetus. This limitation has potential consequences on fetal O2 consumption both in animal models and in human IUGR.